Methods of treating and preventing staphylococcus aureus infections and associated conditions

ABSTRACT

The present invention relates to methods and compositions for preventing and treating Staphylococcus aureus in a subject. Therapeutic compositions of the present invention comprise leukocidin E and/or D proteins or polypeptides and anti-leukocidin E and/or D antibodies. The invention further relates to methods of identifying inhibitors of LukE/D cytotoxicity and inhibitors of LukE/D-leukocyte binding.

This application is a continuation of U.S. patent application Ser. No.15/699,345, filed Sep. 8, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/273,914, filed Sep. 23, 2016, now U.S. Pat. No.9,783,597, issued Oct. 10, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/736,751, filed Jun. 11, 2015, now U.S. Pat. No.9,481,723, issued Nov. 1, 2016, which is a division of U.S. patentapplication Ser. No. 13/527,436, filed Jun. 19, 2012, now U.S. Pat. No.9,091,689, issued on Jul. 28, 2015, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/498,596, filed Jun. 19, 2011,each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods of screening for, treating, andpreventing Staphylococcus aureus infections and Staphylococcus aureusassociated conditions.

BACKGROUND OF THE INVENTION

Staphylococcus aureus (“S. aureus”) is a bacterium that commensallycolonizes more than 25% of the human population. Importantly, thisorganism is capable of breaching its initial site of colonization,resulting in bacterial dissemination and disease. S. aureus is theleading cause of nosocomial infections, is the most common etiologicalagent of infectious endocarditis as well as skin and soft tissueinfections, and is one of the four leading causes of food-borne illness.Altogether, S. aureus infects more than 1.2 million patients per year inU.S. hospitals. The threat of S. aureus to human health is furtherhighlighted by the emergence of antibiotic-resistant strains (i.e.,methicillin-resistant S. aureus (MRSA) strains), including strains thatare resistant to vancomycin, an antibiotic considered the last line ofdefense against S. aureus infection. These facts highlight theimportance of developing novel therapeutics against this importantpathogen.

S. aureus produces a diverse array of virulence factors and toxins thatenable this bacterium to neutralize and withstand attack by differentkinds of immune cells, specifically subpopulations of white blood cellsthat make up the body's primary defense system. The production of thesevirulence factors and toxins allow S. aureus to maintain an infectiousstate (see Nizet, “Understanding How Leading Bacterial Pathogens SubvertInnate Immunity to Reveal Novel Therapeutic Targets,” J. Allergy Clin.Immunol. 120(1):13-22 (2007)). Among these virulence factors, S. aureusproduces several bi-component leukotoxins, which damage membranes ofhost defense cells and erythrocytes by the synergistic action of twonon-associated proteins or subunits (see Menestrina et al., “Mode ofAction of Beta-Barrel Pore-Forming Toxins of the StaphylococcalAlpha-Hemolysin Family,” Toxicol. 39(11):1661-1672 (2001)). Among thesebi-component leukotoxins, gamma-hemolysin (HlgAB and HlgCB) and thePantone-Valentine Leukocidin (PVL) are the best characterized.

The toxicity of the leukocidins towards mammalian cells involves theaction of two components or subunits. The first subunit is named classS-subunit (i.e., “slow-eluted”), and the second subunit is named classF-subunit (i.e., “fast-eluted”). The S- and F-subunits actsynergistically to form pores on white blood cells including monocytes,macrophages, dendritic cells, and neutrophils (collectively known asphagocytes) (see Menestrina et al., “Mode of Action of Beta-BarrelPore-Forming Toxins of the Staphylococcal Alpha-Hemolysin Family,”Toxicol. 39(11):1661-1672 (2001)). The mechanism by which thebi-component toxins form pores in target cell membranes is not entirelyunderstood. The proposed mechanism of action of these toxins involvesbinding of the S-subunit to the target cell membrane, most likelythrough a receptor, followed by binding of the F-subunit to theS-subunit, thereby forming an oligomer which in turn forms a pre-porethat inserts into the target cell membrane (Jayasinghe et al., “TheLeukocidin Pore: Evidence for an Octamer With Four LukF Subunits andFour LukS Subunits Alternating Around a Central Axis,” Protein. Sci.14(10):2550-2561 (2005)). The pores formed by the bi-componentleukotoxins are typically cation-selective. Pore formation causes celldeath via lysis, which in the cases of the target white blood cells, hasbeen reported to result from an osmotic imbalance due to the influx ofcations (Miles et al., “The Staphylococcal Leukocidin Bicomponent ToxinForms Large Ionic Channels,” Biochemistry 40(29):8514-8522 (2001)).

In addition to PVL (also known as leukocidin S/F-PV or LukSF-PV) andgamma-hemolysin (HlgAB and HlgCB), the repertoire of bi-componentleukotoxins produced by S. aureus is known to include leukocidin E/D(“LukE/D”), leukocidin A/B (“LukAB”) and leukocidin M/F (“LukMF”). Thus,the S-class subunits of these bi-component leukocidins include HlgA,HlgC, LukE, LukS-PV, LukA, and LukM, and the F-class subunits includeHlgB, LukD, LukF-PV, LukB, and LukF′-PV. The S. aureus S- and F-subunitsare not leukocidin-specific. That is, they are interchangeable such thatother bi-component combinations could make a functional pore in a whiteblood cell, greatly increasing the repertoire of leukotoxins (Meyer etal., “Analysis of the Specificity of Panton-Valentine Leucocidin andGamma-Hemolysin F Component Binding,” Infect. Immun. 77(1):266-273(2009)).

Designing effective therapy to treat MRSA infection has been especiallychallenging. In addition to the resistance to methicillin and relatedantibiotics, MRSA has also been found to have significant levels ofresistance to macrolides (e.g., erythromycin), beta-lactamase inhibitorcombinations (e.g., Unasyn, Augmentin), and fluoroquinolones (e.g.ciprofloxacin), as well as to clindamycin, trimethoprim/sulfamethoxisol(Bactrim), and rifampin. In the case of serious S. aureus infection,clinicians have resorted to intravenous vancomycin. However, there havebeen reports of S. aureus resistance to vancomycin. Thus, there is aneed to develop new treatments that effectively combat S. aureusinfection.

The present invention is directed to overcoming these and otherlimitations in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a compositioncomprising a therapeutically effective amount of an isolated aLeukocidin E (LukE) protein or polypeptide thereof, an isolatedLeukocidin D (LukD) protein or polypeptide thereof, or a combinationthereof, and a pharmaceutically acceptable carrier.

Another aspect of the present invention relates to a method ofimmunizing against a Staphylococcus aureus infection in a subject. Thismethod involves administering a composition of the present invention inan amount effective to immunize against S. aureus infection in thesubject.

Another aspect of the present invention relates to a compositioncomprising a therapeutically effective amount of an antibody selectedfrom the group consisting of a LukE antibody, a LukD antibody, or acombination thereof, and a pharmaceutically acceptable carrier.

Another aspect of the present invention is directed to a method ofpreventing a S. aureus infection and/or S. aureus-associated conditionsin a subject. This method involves administering a compositioncomprising an antibody selected from the group consisting of a LukEantibody, a LukD antibody, or a combination thereof, in an amounteffective to prevent S. aureus infection and/or S. aureus associatedcondition in the subject.

A further aspect of the present invention is directed to a method oftreating a S. aureus infection and/or S. aureus-associated conditions ina subject. This method involves administering a composition comprisingone or more inhibitors of LukE/D mediated cytotoxicity in an amounteffective to treat the S. aureus infection and/or the S. aureusassociated condition in the subject.

A further aspect of the present invention relates to a method ofpredicting severity of an S. aureus infection. This method involvesculturing S. aureus obtained from an infected subject via a fluid ortissue sample from the subject and quantifying LukE and/or LukDexpression in the cultured S. aureus. The quantified amounts of LukEand/or LukD in the sample from the subject are compared to the amount ofLukE and/or LukD in a control sample which produces little orundetectable amounts of LukE and/or LukD and the severity of the S.aureus infection is predicted based on said comparing.

Another aspect of the present invention relates to a method of treatinga subject with a S. aureus infection. This method involves culturing S.aureus obtained from an infected subject via a fluid or tissue samplefrom the subject and quantifying LukE and/or LukD expression in thecultured S. aureus. The quantified amounts of LukE and/or LukD in thesample from the subject are compared to the amount of LukE and/or LukDin a control sample which produces little or undetectable amounts ofLukE and/or LukD and a suitable treatment for the subject is determinedbased on this comparison. The method further involves administering thedetermined suitable treatment to the subject to treat the S. aureusinfection.

Another aspect of the present invention relates to a method ofidentifying inhibitors of LukE/D cytotoxicity. This method involvesproviding a cell population, a preparation containing LukE/D, and acandidate LukE/D inhibitor. The cell population is exposed to thepreparation containing LukE/D in the presence and absence of thecandidate inhibitor, and LukE/D mediated cytotoxicity is measured in thepresence and in the absence of the candidate inhibitor. The measuredamount of cytotoxicity in the presence and in the absence of thecandidate inhibitor is compared and an inhibitor of LukE/D cytotoxicityis identified based on that comparison.

Another aspect of the present invention relates to a method ofidentifying inhibitors of LukE/D mediated pore formation. This methodinvolves providing a population of leukocytes, a preparation containingLukE and LukD, and a candidate inhibitor. The leukocyte population isexposed to the preparation containing LukE and LukD in the presence andabsence of the candidate inhibitor, and pore formation on the leukocytepopulation is measured in the presence and absence of the candidateinhibitor. The measured amount of pore formation in the presence and inthe absence of the candidate inhibitor is compared, and an inhibitor ofLukE/D mediated pore formation is identified based on that comparison.

Another aspect of the present invention is directed to a method ofidentifying inhibitors of LukE and/or LukD leukocyte binding. Thismethod involves providing a population of leukocytes, a preparationcontaining a detectably labeled LukE and LukD, and a candidateinhibitor. The cell population is exposed to the preparation containingthe detectably labeled LukE and LukD in the presence and absence of thecandidate inhibitor, and labeled LukE and/or LukD binding to theleukocyte population is measured in the presence and absence of thecandidate inhibitor. The measured amount of LukE and/or LukD leukocytebinding in the presence and in the absence of the candidate inhibitor iscompared and an inhibitor of LukE and/or LukD leukocyte binding isidentified based on that comparison.

The tremendous success of S. aureus as a pathogen is in part due to itsability to express an arsenal of factors that harm the host. Among thesefactors are a number of bacterial protein toxins that are secreted intothe extracellular milieu where they act by killing host cells.Leukocidin E/D (LukE/D) is a poorly characterized toxin produced by S.aureus. As demonstrated herein, this toxin targets and kills hostleukocytes, which are key immune cells involved in protecting the hostfrom S. aureus infection. The finding that LukE/D is critical topathogenesis in vivo, highlights the importance of this toxin in thedisease process. As described herein, immunization with LukE and/or LukDgenerates neutralizing antibodies against S. aureus. Therefore, activeand/or passive vaccine strategies offer a novel therapeutic strategy toprevent S. aureus infection. In addition, direct inhibition of LukE/Dmeditated cytotoxicity offers a novel means of treating individuals withS. aureus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show that deletion of the rot gene in an S. aureus lackingthe agr locus (ΔagrΔrot) restores virulence in mice to wild type (“WT”)levels and leads to overproduction of LukE/D. FIG. 1A is a survivalcurve showing that an Δagr Δrot double mutant exhibits WT virulencecharacteristics in mice. Survival of mice was monitored afterintravenous injection with ˜1×10⁷ CFU of S. aureus WT, Δagr, or ΔagrΔrot double mutants. Total number of mice per group were N=6.Statistical significance between curves was determined using theLog-rank (Mantel-Cox) test. ***, p≤0.0005. In FIG. 1B, the production ofleukotoxins is restored in an Δagr Δrot double mutant. Shown areimmunoblots of protein samples from TCA precipitated bacterial culturesupernatants (grown for 5 hours in RPMI+CAS) of the following strains:WT, Δagr, and Δagr Δrot. Negative control lanes contain TCA precipitatedsupernatant from respective leukotoxin deletion mutants (ΔlukE/D,ΔlukA/B, Δhla, ΔhlgC). ΔlukE/D ΔhlgACB double mutant exoproteins werealso probed in all the LukE immunoblots as a control for LukE antibodycross-reactivity.

FIGS. 2A-2C illustrate that deletion of rot alone results inhypervirulence in animals, a phenotype caused by derepression andresultant overproduction of LukE/D. The survival curve of FIG. 2A showsthe hypervirulence of a Δrot mutant compared to the parent WT strain.Survival of mice was monitored after intravenous injection with ˜1×10⁷CFU of S. aureus WT and Δrot strains. Total number of mice per group:WT, N=17; Δrot, N=12. The production of LukE/D is increased in theabsence of the transcriptional repressor Rot, while the production ofother leukotoxins is largely unaffected. Shown in the immunoblots ofFIG. 2B are protein samples from TCA precipitated bacterial culturesupernatants (grown for 5 hours in RPMI+CAS) of the following strains:WT, and Δrot. Negative control lanes contain TCA precipitatedsupernatant from respective toxin-rot double mutants (Δrot ΔlukE/D, ΔrotΔlukA/B, Δrot Δhla, and Δrot ΔhlgACB). Δrot ΔlukE/D ΔhlgACB triplemutant exoproteins were also probed in all the LukE immunoblots as acontrol for LukE antibody cross-reactivity. As indicated by the survivalcurve of FIG. 2C, the hypervirulence of a Δrot mutant is due toincreased production of LukE/D. Survival of mice was monitored afterintravenous injection with ˜1×10⁷ CFU of S. aureus WT, Δrot, and ΔrotΔlukE/D. Statistical significance between survival curves was determinedusing the Log-rank (Mantel-Cox) test. **, p≤0.005; ***, p≤0.0005.

FIGS. 3A-3B show that Rot binds to the lukE/D promoter and repressesgene expression. As shown in FIG. 3A, optimal lukE/D gene expression isdependent on derepression of Rot. Transcriptional fusions of the lukE/Dpromoter region to GFP were used to measure activation of the promoterin broth culture in the following strain backgrounds (WT, Δagr, Δrot,and Δagr Δrot). GFP fluorescence was measured over time and valuesexpressed as relative fluorescent units (RFU) after normalization tobacterial Optical Density at 600 nm. Values shown are results of threeexperiments performed in triplicate. In FIG. 3B, Rot binds to the lukE/Dpromoter. FIG. 3B is an immunoblot of a promoter pull-down of eitherbiotinylated intragenic DNA (non-specific) or lukE/D promoter DNA boundto M280 streptavidin magnetic beads and incubated with S. aureus wholecell lysates. Rot was detected via immunoblot using an anti-Rotantibody.

FIGS. 4A-4F illustrate that a ΔlukE/D single mutant is significantlyattenuated for virulence in a mouse model of systemic infection. FIGS.4A and 4B show verification of the lukE/D deletion in S. aureus Newman.In FIG. 4A, PCR of S. aureus genomic DNA with lukE specific primers isshown. Shown in FIG. 4B are immunoblots of protein samples from TCAprecipitated bacterial culture supernatants (grown for 5 hours inRPMI+CAS) of the following strains: WT, ΔlukE/D, ΔlukE/D::plukE/D,ΔhlgACB, and ΔhlgACB. ΔlukE/D mutant exoproteins were also probed as acontrol for LukE antibody cross-reactivity. FIGS. 4C-4F show thatΔlukE/D mutant is severely compromised for virulence in mice. In FIGS.4C and 4D, the survival of mice was monitored after intravenousinjection with ˜1×10⁷ CFU (FIG. 4C) or ˜1×10⁸ CFU (FIG. 4D) of S. aureusWT, ΔlukE/D, and ΔlukE/D::plukE/D strains. Total number of mice pergroup were N=6. Statistical significance between survival curves wasdetermined using the Log-rank (Mantel-Cox) test. **, p≤0.005; ***,p≤0.0005. FIGS. 4E and 4F depict enumeration of bacterial CFU (FIG. 4E)and gross pathology (FIG. 4F) from kidneys 96 hours post-infection with˜1×10⁷ CFU of the same strains described for FIGS. 4C and 4D. Arrowsdesignate locations of kidney abscesses. Statistical significance wasdetermined using 1-Way ANOVA with Tukey's multiple comparisons posttest.**, p≤0.005; ***, p≤0.0005.

FIGS. 5A-5E show that LukE/D is toxic to and forms pores in human immunecells. FIG. 5A is a cell viability curve showing that purifiedrecombinant LukE/D is toxic to the human monocyte-like cell line THP-1.The THP-1 cell line was intoxicated with recombinant LukE, LukD, or amixture of LukE+LukD (LukE/D). Cell viability was monitored 1 hourpost-intoxication using CellTiter, where cells treated with medium wereset at 100% viable. Results represent the average of triplicatesamples±S.D. Purified recombinant LukE/D is not toxic to the human HL60cell line, as shown in the cell viability curve of FIG. 5B. The HL60cell line was intoxicated as above and cell viability was monitored 1hour post-intoxication using CellTiter, where cells treated with mediumwere set at 100% viable. In contrast, the cell viability curves of FIG.5C show purified recombinant LukE/D is toxic to both primary human (leftgraph) and primary murine (right graph) neutrophils (also known aspolymorphonuclear neutrophils or PMNs). The PMNs were intoxicated asabove and cell viability was monitored 1 hour post-intoxication usingCellTiter, where cells treated with medium were set at 100% viable.LukE/D mediates cytotoxicity toward host cells THP-1 cells by formingpores in the cell membrane as shown in FIG. 5D. THP-1 and HL60 cellswere incubated with purified LukE/D, and pore formation was measuredwith an ethidium bromide incorporation assay. Mean fluorescence oftriplicate experiments are shown for both THP-1 and HL60. FIG. 5E showsa fluorescence microscopy image of ethidium bromide uptake of LukE/Dtreated (30 μg/ml) and control (no toxin) THP-1 cells.

FIGS. 6A-6B illustrate that LukE/D cytotoxicity is neutralized with anaffinity purified α-LukE polyclonal antibody. THP-1 cells wereintoxicated with 1.5 μg of recombinant LukE/D following incubation with0.1 μg α-LukE polyclonal antibody or pre-immune serum. Cell viability(FIG. 6A) and pore formation (FIG. 6B) were monitored using CellTiterand Ethidium bromide respectively. For CellTiter assays, cells treatedwith medium were set at 100% viability. Results represent the average ofduplicate samples±standard deviation (S.D.).

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a compositioncomprising a therapeutically effective amount of an isolated LukEprotein or polypeptide thereof, an isolated LukD protein or polypeptidethereof, or a combination thereof, and a pharmaceutically acceptablecarrier.

In one embodiment of the invention, the composition comprises anisolated LukE protein or polypeptide. In another embodiment of theinvention, the composition comprises an isolated LukD protein orpolypeptide. In yet another embodiment of the invention the compositioncomprises both LukE and LukD proteins or polypeptides.

In accordance with this aspect of the invention, suitable isolated LukEproteins include those derived from any strain of S. aureus. The aminoacid sequence of LukE proteins from various strains of S. aureus thatare suitable for the composition of the present invention are shown inthe Table 1 below (i.e., SEQ ID Nos:1-10). SEQ ID NO:11 of Table 1 is aLukE consensus sequence demonstrating the high level of sequenceidentity across LukE proteins of various S. aureus strains. Accordingly,in one embodiment of the present invention, the isolated LukE proteincomprises an amino acid sequence of SEQ ID NO:11. In another embodimentof the present invention, the isolated LukE protein comprises an aminoacid sequence having about 70-80% sequence similarity to SEQ ID NO:11,more preferably, about 80-90% sequence similarity to SEQ ID NO:11, andmore preferably 90-95% sequence similarity to SEQ ID NO:11, and mostpreferably about 95-99% sequence similarity to SEQ ID NO:11.

In another embodiment of the present invention, the compositioncomprises an isolated immunogenic polypeptide of LukE. Suitable LukEpolypeptides are about 50 to about 100 amino acids in length. Morepreferably LukE polypeptides are between about 100-200 amino acids inlength, more preferably between about 200-250 amino acids in length, andmost preferably between 250-300 amino acids in length. The N-terminalamino acid residues of the full-length LukE represent the nativesecretion/signal sequence. Thus, the “mature” secreted form of LukE isrepresented by amino acid residues 29-311 in each of SEQ ID NOs:1-10 andSEQ ID NO:11. Correspondingly, amino acid residues 1-311 in each of SEQID NOs:1-10 and SEQ ID NO:11 are referred to as the “immature” form ofLukE. Accordingly, in one embodiment of the present invention, the LukEpolypeptide comprises amino acid residues 29-311 of SEQ ID NO:11.Alternatively, the LukE polypeptide of the present invention comprisesamino acid residues 48-291, amino acids 29-301, or amino acids 48-301 ofSEQ ID NO:11. These LukE polypeptides lack LukE activity but maintainantigenicity. In either case, suitable LukE polypeptides also includethose polypeptides comprising an amino acid sequence having about 70-80%sequence similarity, preferably 80-90% sequence similarity, morepreferably 90-95% sequence similarity, and most preferably 95-99%sequence similarity to amino acid residues 29-311 of SEQ ID NO:11, aminoacid residues 48-291 of SEQ ID NO:11, amino acid residues 29-301 of SEQID NO:11, or amino acid residues 48-301 of SEQ ID NO:11.

In accordance with this aspect of the invention, suitable isolated LukDproteins include those proteins derived from any strain of S. aureus.The amino acid sequence of LukD proteins from various strains of S.aureus that are suitable for the composition of the present inventionare shown in the Table 2 below (i.e., SEQ ID Nos: 12-21). SEQ ID NO:22of Table 2 is a LukD consensus sequence demonstrating the high level ofsequence identity across LukD proteins of various S. aureus strains.Accordingly, in one embodiment of the present invention, the isolatedLukD protein comprises an amino acid sequence of SEQ ID NO:22. Inanother embodiment of the present invention, the isolated LukD proteincomprises an amino acid sequence having about 70-80% sequence similarityto SEQ ID NO:22, preferably, about 80-90% sequence similarity to SEQ IDNO:22, and more preferably 90-95% sequence similarity to SEQ ID NO:22,and most preferably about 95-99% sequence similarity to SEQ ID NO:22.

In another embodiment of the present invention, the compositioncomprises an isolated immunogenic polypeptide of LukD. Suitable LukDpolypeptides are about 50 to about 100 amino acids in length. Morepreferably LukD polypeptides are between about 100-200 amino acids inlength, more preferably between about 200-250 amino acids in length, andmost preferably between 250-300 amino acids in length. The N-terminalamino acid residues of the full-length LukD represent the nativesecretion/signal sequence. Thus, the mature secreted form of LukD isrepresented by amino acid residues 27-327 in each of SEQ ID NOs:12-21and SEQ ID NO:22. Correspondingly, amino acid residues 1-327 of SEQ IDNOs:12-21 and SEQ ID NO:22 are referred to as the “immature” form ofLukD. Accordingly, in one embodiment of the present invention, the LukEpolypeptide comprises amino acid residues 27-327 of SEQ ID NO:22.Alternatively, the LukE polypeptide of the present invention comprisesamino acid residues 46-307, 27-312, and 46-312 of SEQ ID NO:22. TheseLukD polypeptide lack LukD activity but maintain antigenicity. In eithercase, suitable polypeptides also include those polypeptide comprising anamino acid sequence having about 70-80% sequence similarity, preferably80-90% sequence similarity, more preferably 90-95% sequence similarity,and most preferably 95-99% sequence similarity to amino acid residues27-327 of SEQ ID NO:22, amino acid residues 46-307 of SEQ ID NO:22,amino acid residues 27-312 of SEQ ID NO:22, or amino acid residues46-312 of SEQ ID NO:22.

TABLE 1 S. Aureus LukE Sequence Alignment S. Aureus Strain                             → NewmanMFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50 SEQ ID NO: 1 MW2MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50 SEQ ID NO: 2USA_300_FPR3757 MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50SEQ ID NO: 3 COL MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50SEQ ID NO: 4 USA_300_TCH1516MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50 SEQ ID NO: 5 N315MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50 SEQ ID NO: 6 D30MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50 SEQ ID NO: 7 Mu50MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50 SEQ ID NO: 8TCH_70 MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50SEQ ID NO: 9 MRSA131 MFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS 50 SEQ ID NO: 10 **************************************************LukE Consensus SequenceMFKKKMLAATLSVGLIAPLASPIQESRANTNIENIGDGAEVIKRTEDVSS  50 SEQ ID NO: 11Newman KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 MW2KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 USA_300_FPR3757KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 COLKKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 USA_300_TCH1516KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 N315KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 D30KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 Mu50KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 TCH_70KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100 MRSA131KKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK 100**************************************************LukE Consensus SequenceKKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTK NewmanRMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 MW2RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 USA_300_FPR3757RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 COLRMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 USA_300_TCH1516RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 N315RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 D30RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 Mu50RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 TCH_70RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150 MRSA131RMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA 150**************************************************LukE Consensus SequenceRMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSA NewmanPSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 MW2PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 USA_300_FPR3757PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 COLPSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 USA_300_TCH1516PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 N315PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 D30PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 Mu50PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 TCH_70PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200 MRSA131PSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK 200**************************************************LukE Consensus SequencePSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKK NewmanSAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 MW2SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 USA_300_FPR3757SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 COLSAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 USA_300_TCH1516SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 N315SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 D30SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 Mu50SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 TCH_70SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250 MRSA131SAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG 250**************************************************LukE Consensus SequenceSAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKG NewmanSSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 MW2SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 USA_300_FPR3757SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 COLSSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 USA_300_TCH1516SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 N315SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 D30SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 Mu50SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 TCH_70SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300 MRSA131SSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW 300**************************************************LukE Consensus SequenceSSDTSEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFVVRYEVNW Newman KTHEIKVKGHN311 MW2 KTHEIKVKGHN 311 USA_300_FPR3757 KTHEIKVKGHN 311 COL KTHEIKVKGHN311 USA_300_TCH1516 KTHEIKVKGHN 311 N315 KTHEIKVKGHN 311 D30 KTHEIKVKGHN311 Mu50 KTHEIKVKGHN 311 TCH_70 KTHEIKVKGHN 311 MRSA131 KTHEIKVKGHN 311*********** LukE Consensus Sequence KTHEIKVKGHN →Depicts the start ofthe secreted LukE protein

TABLE 2 LukD Amino Acid Sequence Alignment                            →Newman MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50SEQ ID NO: 12 MW2 MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50SEQ ID NO: 13 USA_300_FPR3757MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50 SEQ ID NO: 14 COLMKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50 SEQ ID NO: 15USA_300_TCH1516 MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50SEQ ID NO: 16 MRSA131 MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT50 SEQ ID NO: 17 TCH_70MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50 SEQ ID NO: 18 D30MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50 SEQ ID NO: 19 N315MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50 SEQ ID NO: 20 Mu50MKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50 SEQ ID NO: 21**************************************************LukD Consensus SequenceMKMKKLVKSSVASSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTAT 50 SEQ ID NO: 22Newman SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 MW2SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 USA_300_FPR3757SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 COLSDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 USA_300_TCH1516SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 MRSA131SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 TCH_70SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 D30SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 N315SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100 Mu50SDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS 100**************************************************LukD Consensus SequenceSDNDKLNISQILTENFIKDKSYDKDTLVLKAAGNINSGYKKPNPKDYNYS NewmanQFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 MW2QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 USA_300_FPR3757QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 COLQFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 USA_300_TCH1516QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 MRSA131QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 TCH_70QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 D30QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 N315QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150 Mu50QFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI 150**************************************************LukD Consensus SequenceQFYWGGKYNVSVSSESNDAVNVVDTAPKNQNEEFQVQQTLGYSYGGDINI NewmanSNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 MW2SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 USA_300_FPR3757SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 COLSNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 USA_300_TCH1516SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 MRSA131SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 TCH_70SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 D30SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 N315SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200 Mu50SNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN 200**************************************************LukD Consensus SequenceSNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNN NewmanGWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 MW2GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 USA_300_FPR3757GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 COLGWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 USA_300_TCH1516GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 MRSA131GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 TCH_70GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 D30GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 N315GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250 Mu50GWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE 250**************************************************LukD Consensus SequenceGWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNPE NewmanFISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 MW2FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 USA_300_FPR3757FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 COLFISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 USA_300_TCH1516FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 MRSA131FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 TCH_70FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 D30FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTF 300 N315FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWIGNNYKNQNTVTF 300 Mu50FISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWIGNNYKNQNTVTF 300*************************************:************LukD Consensus SequenceFISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWXGNNYKNQNTVTF NewmanTSTYEVDWQNHTVKLIGTDSKETNPGV 327 MW2 TSTYEVDWQNHTVKLIGTDSKETNPGV 327USA_300_FPR3757 TSTYEVDWQNHTVKLIGTDSKETNPGV 327 COLTSTYEVDWQNHTVKLIGTDSKETNPGV 327 USA_300_TCH1516TSTYEVDWQNHTVKLIGTDSKETNPGV 327 MRSA131 TSTYEVDWQNHTVKLIGTDSKETNPGV 327TCH_70 TSTYEVDWQNHTVKLIGTDSKETNPGV 327 D30 TSTYEVDWQNHTVKLIGTDSKETNPGV327 N315 TSTYEVDWQNHTVKLIGTDSKETNPGV 327 Mu50TSTYEVDWQNHTVKLIGTDSKETNPGV 327 ***************************LukD Consensus Sequence TSTYEVDWQNHTVKLIGTDSKETNPGV →Depicts the startof the secreted LukD protein

Thus, unless indicated to the contrary, both the immature and the matureforms of native LukE and LukD, and the sequences having less than 100%similarity with native LukE and LukD (i.e., native sequences and analogsalike, collectively referred to herein as “LukE” and “LukD”) may be usedin the methods of the present invention.

LukE and LukD proteins and polypeptides of the invention may differ fromthe native polypeptides designated as SEQ ID NOS:1-11 and 12-22respectively, in terms of one or more additional amino acid insertions,substitutions or deletions, e.g., one or more amino acid residues withinSEQ ID NOS:1-22 may be substituted by another amino acid of a similarpolarity, which acts as a functional equivalent, resulting in a silentalteration. That is to say, the change relative to the native sequencewould not appreciably diminish the basic properties of native LukE orLukD. Any such analog of LukE or LukD may be screened in accordance withthe protocols disclosed herein (e.g., the cell toxicity assay and themembrane damage assay) to determine if it maintains native LukE or LukDactivity. Substitutions within these leukocidins may be selected fromother members of the class to which the amino acid belongs. For example,nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. Polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. Positively charged (basic) amino acidsinclude arginine, lysine and histidine. Negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

In other embodiments, non-conservative alterations (e.g., one or aminoacid substitutions, deletions and/or additions) can be made for purposesof detoxifying LukE and/or LukD. The detoxified LukE and LukD may beused in the active vaccine compositions. Molecular alterations can beaccomplished by methods well known in the art, including primerextension on a plasmid template using single stranded templates (Kunkelet al., Proc. Acad. Sci., USA 82:488-492 (1985), which is herebyincorporated by reference in its entirety), double stranded DNAtemplates (Papworth, et al., Strategies 9(3):3-4 (1996), which is herebyincorporated by reference in its entirety), and by PCR cloning (Braman,J. (ed.), IN VITRO MUTAGENESIS PROTOCOLS, 2nd ed. Humana Press, Totowa,N.J. (2002), which is hereby incorporated by reference in its entirety).Methods of determining whether a given molecular alteration in LukE andLukD reduces LukE/D cytotoxicity are described herein.

In a preferred embodiment of the present invention, a highly purifiedLukE/LukD preparation is utilized. Examples include LukE and LukDproteins or polypeptides purified from the various strains exemplifiedin Tables 1 and 2. Methods of purifying LukE and LukD toxins are knownin the art (Gravet et al., “Characterization of a Novel StructuralMember, LukE-LukD, of the Bi-Component Staphylococcal LeucotoxinsFamily,” FEBS 436: 202-208 (1998), which is hereby incorporated byreference in its entirety). As used herein, “isolated” protein orpolypeptide refers to a protein or polypeptide that has been separatedfrom other proteins, lipids, and nucleic acids with which it isnaturally associated with. Purity can be measured by any appropriatestandard method, for example, by column chromatography, polyacrylamidegel electrophoresis, of HPLC analysis. An isolated protein orpolypeptide of the invention can be purified from a natural source,produced by recombinant DNA techniques, or by chemical methods.

In one embodiment of this aspect of the present invention, the isolatedLukE or LukD protein or polypeptide thereof of the composition is linkedto an immunogenic carrier molecule. In some cases, the immunogeniccarrier molecule may be covalently or non-covalently bound to theimmunogenic protein or peptide. Exemplary immunogenic carrier moleculesinclude, but are not limited to, bovine serum albumin, chicken eggovalbumin, keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid,thyro globulin, a pneumococcal capsular polysaccharide, CRM 197, and ameningococcal outer membrane protein.

In certain embodiments of the present invention, the composition mayfurther contain one or more additional S. aureus antigens. Suitable S.aureus antigens include, without limitation, alpha hemolysin antigen,protein A, a serotype 336 polysaccharide antigen, coagulase, clumpingfactor A, clumping factor B, a fibronectin binding protein, a fibrinogenbinding protein, a collagen binding protein, an elastin binding protein,a MEW analogous protein, a polysaccharide intracellular adhesion, betahemolysin, delta hemolysin, gamma hemolysin, Panton-Valentineleukocidin, leukocidin A, leukocidin B, leukocidin M, exfoliative toxinA, exfoliative toxin B, V8 protease, hyaluronate lyase, lipase,staphylokinase, an enterotoxin, toxic shock syndrome toxin-1,poly-N-succinyl beta-1→6 glucosamine, catalase, beta-lactamase, teichoicacid, peptidoglycan, a penicillin binding protein, chemotaxis inhibitingprotein, complement inhibitor, Sbi, Type 5 antigen, Type 8 antigen,lipoteichoic acid, and microbial surface proteins that recognize hostproteins (e.g., iron surface determinents, serine-aspartate repeatproteins).

In accordance with this aspect of the invention, the composition mayfurther comprise one or more adjuvants. Suitable adjuvants are known inthe art and include, without limitation, flagellin, Freund's complete orincomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols,polyanions, peptides, oil emulsion, dinitrophenol, iscomatrix, andliposome polycation DNA particles.

In embodiments wherein the therapeutic composition is intended for useas an active vaccine, the LukE and/or LukD proteins or polypeptides maybe altered so as to exhibit reduced toxicity. Alterations for purposesof reducing toxicity of LukE and LukD include chemical treatment (e.g.,modification of specific amino acid residues as described supra) orconjugation to another moiety (e.g., to another bacterial antigen, suchas a bacterial polysaccharide or a bacterial glycoprotein). Chemicalalterations to other S. aureus toxins for purposes of detoxification (orreducing toxicity) are known. Methods of determining whether a givenalteration reduces LukE or LukD toxicity are known in the art and/ordescribed herein.

The therapeutic compositions of the present invention are prepared byformulating LukE and LukD with a pharmaceutically acceptable carrier andoptionally a pharmaceutically acceptable excipient. As used herein, theterms “pharmaceutically acceptable carrier” and “pharmaceuticallyacceptable excipient” (e.g., additives such as diluents,immunostimulants, adjuvants, antioxidants, preservatives andsolubilizing agents) are nontoxic to the cell or mammal being exposedthereto at the dosages and concentrations employed. Examples ofpharmaceutically acceptable carriers include water, e.g., buffered withphosphate, citrate and another organic acid. Representative examples ofpharmaceutically acceptable excipients that may be useful in the presentinvention include antioxidants such as ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; adjuvants (selected so as toavoid adjuvant-induced toxicity, such as a β-glucan as described in U.S.Pat. No. 6,355,625, which is hereby incorporated by reference in itsentirety, or a granulocyte colony stimulating factor (GCSF));hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

Therapeutic compositions of the present invention may be prepared forstorage by mixing the active ingredient(s) having the desired degree ofpurity with the pharmaceutically acceptable carrier and optionalexcipient and/or additional active agent, in the form of lyophilizedformulations or aqueous solutions.

Another aspect of the present invention relates to a method ofimmunizing against a Staphylococcus aureus infection in a subject. Thismethod involves administering a composition of the present invention, inan amount effective to immunize against S. aureus infection in thesubject. A suitable subject for treatment in accordance with this aspectof the present invention is a subject at risk of developing a S. aureusinfection.

In accordance with this aspect of the invention, a therapeuticallyeffective amount of the composition for administration to a subject toimmunize against S. aureus infection is the amount necessary to generatea humoral (i.e., antibody mediated) immune response. Preferably,administration of a therapeutically effective amount of the compositionof the present invention induces a neutralizing immune response againstS. aureus in the subject. To effectuate an effective immune response ina subject, the composition may further contain one or more additional S.aureus antigens or an adjuvant as described supra. In an alternativeembodiment of this aspect of the invention, the adjuvant is administeredseparately from the composition to the subject, either before, after, orconcurrent with administration of the composition of the presentinvention.

Modes of administration and therapeutically effective dosing related tothis aspect of the invention are described infra.

Another aspect of the present invention relates to a compositioncomprising a therapeutically effective amount of an antibody selectedfrom the group consisting of a Leukocidin E (LukE) antibody, aLeukocidin D (LukD) antibody, or a combination thereof, and apharmaceutically acceptable carrier.

In one embodiment of this aspect of the present invention, thecomposition comprises a LukE antibody or antigen-binding fragmentthereof. Suitable LukE antibodies include those antibodies recognizingone or more epitopes in the amino acid sequence of SEQ ID NO:11.Likewise, in another embodiment, the composition comprises a LukDantibody or antigen-binding fragment thereof. Suitable LukD antibodiesrecognize one or more epitopes in the amino acid sequence of SEQ IDNO:22. In another embodiment of the invention, the composition comprisesboth LukE and LukD antibodies or antigen binding fragments thereof.Preferably, the composition comprises one or more neutralizing LukEand/or LukD antibodies. In yet another embodiment, the anti-LukE and/oranti-LukD antibody composition is multivalent in that it also containsan antibody that specifically binds another bacterial antigen (and thatoptionally neutralizes the other bacterial antigen). For example, thecomposition may comprise one or more antibodies that recognize one ormore additional S. aureus antigens, including, without limitation, oneor more of the S. aureus antigens described supra.

For purposes of the present invention, the term “antibody” includesmonoclonal antibodies, polyclonal antibodies, antibody fragments,genetically engineered forms of the antibodies, and combinationsthereof. More specifically, the term “antibody,” which is usedinterchangeably with the term “immunoglobulin,” includes full length(i.e., naturally occurring or formed by normal immunoglobulin genefragment recombinatorial processes) immunoglobulin molecules (e.g., anIgG antibody) and immunologically active fragments thereof (i.e.,including the specific binding portion of the full-length immunoglobulinmolecule), which again may be naturally occurring or synthetic innature. Accordingly, the term “antibody fragment” includes a portion ofan antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv and the like.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the full-length antibody, and, in thecontext of the present invention, specifically binds LukE, LukD, or aLukE/D complex. Methods of making and screening antibody fragments arewell-known in the art.

In the present invention, the anti-LukE antibodies may have some degreeof cross-reactivity with other Staphylococcus leukocidin S-subunits suchas HlgC, LukS-PVL, HlgA, LukS-I, LukA, and LukM. Likewise, in someembodiments, the anti-LukD antibodies of the present invention may havesome degree of cross-reactivity with other Staphylococcus leukocidinF-subunits such as LukF′-PV, LukF-PV, LukB, LukF-I, and HlgB. Anti-LukEand/or anti-LukD antibodies may inhibit or reduce LukE activity and LukDactivity, respectively. In some embodiments, the anti-LukE and/oranti-LukD antibodies neutralize (e.g., substantially eliminate) LukE andLukD activity, respectively.

Naturally occurring antibodies typically have two identical heavy chainsand two identical light chains, with each light chain covalently linkedto a heavy chain by an inter-chain disulfide bond and multiple disulfidebonds further link the two heavy chains to one another. Individualchains can fold into domains having similar sizes (110-125 amino acids)and structures, but different functions. The light chain can compriseone variable domain (VL) and/or one constant domain (CL). The heavychain can also comprise one variable domain (VH) and/or, depending onthe class or isotype of antibody, three or four constant domains (CHI,CH 2, CH3 and CH4). In humans, the isotypes are IgA, IgD, IgE, IgG, andIgM, with IgA and IgG further subdivided into subclasses or subtypes(IgA1-2 and IgG1-4).

Generally, the variable domains show considerable amino acid sequencevariability from one antibody to the next, particularly at the locationof the antigen-binding site. Three regions, called hyper-variable orcomplementarity-determining regions (CDRs), are found in each of VL andVH, which are supported by less variable regions called frameworkvariable regions. The inventive antibodies include IgG monoclonalantibodies as well as antibody fragments or engineered forms. These are,for example, Fv fragments, or proteins wherein the CDRs and/or variabledomains of the exemplified antibodies are engineered as single-chainantigen-binding proteins.

The portion of an antibody consisting of the VL and VH domains isdesignated as an Fv (Fragment variable) and constitutes theantigen-binding site. A single chain Fv (scFv or SCA) is an antibodyfragment containing a VL domain and a VH domain on one polypeptidechain, wherein the N terminus of one domain and the C terminus of theother domain are joined by a flexible linker. The peptide linkers usedto produce the single chain antibodies are typically flexible peptides,selected to assure that the proper three-dimensional folding of the VLand VH domains occurs. The linker is generally 10 to 50 amino acidresidues, and in some cases is shorter, e.g., about 10 to 30 amino acidresidues, or 12 to 30 amino acid residues, or even 15 to 25 amino acidresidues. An example of such linker peptides includes repeats of fourglycine residues followed by a serine residue.

Single chain antibodies lack some or all of the constant domains of thewhole antibodies from which they are derived. Therefore, they canovercome some of the problems associated with the use of wholeantibodies. For example, single-chain antibodies tend to be free ofcertain undesired interactions between heavy-chain constant regions andother biological molecules. Additionally, single-chain antibodies areconsiderably smaller than whole antibodies and can have greaterpermeability than whole antibodies, allowing single-chain antibodies tolocalize and bind to target antigen-binding sites more efficiently.Furthermore, the relatively small size of single-chain antibodies makesthem less likely to provoke an unwanted immune response in a recipientthan whole antibodies.

Fab (Fragment, antigen binding) refers to the fragments of the antibodyconsisting of the VL, CL, VH, and CH1 domains. Those generated followingpapain digestion simply are referred to as Fab and do not retain theheavy chain hinge region. Following pepsin digestion, various Fabsretaining the heavy chain hinge are generated. Those fragments with theinterchain disulfide bonds intact are referred to as F(ab′)₂, while asingle Fab′ results when the disulfide bonds are not retained. F(ab′)₂fragments have higher avidity for antigen that the monovalent Fabfragments.

Fc (Fragment crystallization) is the designation for the portion orfragment of an antibody that comprises paired heavy chain constantdomains. In an IgG antibody, for example, the Fc comprises CH2 and CH3domains. The Fc of an IgA or an IgM antibody further comprises a CH4domain. The Fc is associated with Fc receptor binding, activation ofcomplement mediated cytotoxicity and antibody-dependentcellular-cytotoxicity (ADCC). For antibodies such as IgA and IgM, whichare complexes of multiple IgG-like proteins, complex formation requiresFc constant domains.

Finally, the hinge region separates the Fab and Fc portions of theantibody, providing for mobility of Fabs relative to each other andrelative to Fc, as well as including multiple disulfide bonds forcovalent linkage of the two heavy chains.

Antibody “specificity” refers to selective recognition of the antibodyfor a particular epitope of an antigen. The term “epitope” includes anyprotein determinant capable of specific binding to an immunoglobulin orT-cell receptor or otherwise interacting with a molecule. Epitopicdeterminants generally consist of chemically active surface groupings ofmolecules such as amino acids or carbohydrate or sugar side chains andgenerally have specific three dimensional structural characteristics, aswell as specific charge characteristics. An epitope may be “linear” or“conformational”. In a linear epitope, all of the points of interactionbetween the protein and the interacting molecule (such as an antibody)occur linearly along the primary amino acid sequence of the protein. Ina conformational epitope, the points of interaction occur across aminoacid residues on the protein that are separated from one another, i.e.,noncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents, whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen.

Monoclonal antibodies of the present invention may be murine, human,humanized or chimeric. A humanized antibody is a recombinant protein inwhich the CDRs of an antibody from one species; e.g., a rodent, rabbit,dog, goat, horse, or chicken antibody (or any other suitable animalantibody), are transferred into human heavy and light variable domains.The constant domains of the antibody molecule are derived from those ofa human antibody. Methods for making humanized antibodies are well knownin the art. Chimeric antibodies preferably have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region from a mammal other than a human. Thechimerization process can be made more effective by also replacing thevariable regions—other than the hyper-variable regions or thecomplementarity—determining regions (CDRs), of a murine (or othernon-human mammalian) antibody with the corresponding human sequences.The variable regions other than the CDRs are also known as the variableframework regions (FRs). Yet other monoclonal antibodies of the presentinvention are bi-specific, in that they have specificity for both LukEand LukD. Bispecific antibodies are preferably human or humanized.

The above-described antibodies can be obtained in accordance withstandard techniques. For example, LukE, LukD, or an immunologicallyactive fragment of LukE or LukD can be administered to a subject (e.g.,a mammal such as a human or mouse). The leukocidins can be used bythemselves as immunogens or they can be attached to a carrier protein orother objects, such as sepharose beads. After the mammal has producedantibodies, a mixture of antibody producing cells, such as splenocytes,are isolated, from which polyclonal antibodies may be obtained.Monoclonal antibodies may be produced by isolating individualantibody-producing cells from the mixture and immortalizing them by, forexample, fusing them with tumor cells, such as myeloma cells. Theresulting hybridomas are preserved in culture and the monoclonalantibodies are harvested from the culture medium.

Another aspect of the present invention is directed to a method ofpreventing a S. aureus infection and/or S. aureus-associated conditionsin a subject. This method comprises administering a composition of theinvention comprising an antibody selected from the group consisting of aLeukocidin E (LukE) antibody, a Leukocidin D (LukD) antibody, or acombination thereof, in an amount effective to prevent S. aureusinfection and/or S. aureus associated condition in the subject.

In accordance with this aspect of the invention, S. aureus-associatedconditions include, without limitation, skin wounds and infections,tissue abscesses, folliculitis, osteomyelitis, pneumonia, scalded skinsyndrome, septicemia, septic arthritis, myocarditis, endocarditis, andtoxic shock syndrome.

Modes of administration and therapeutically effective dosing related tothis aspect of the invention are described infra.

A further aspect of the present invention is directed to a method oftreating a S. aureus infection and/or S. aureus-associated conditions ina subject. This method involves administering a composition comprisingone or more inhibitors of LukE/D mediated cytotoxicity in an amounteffective to treat the S. aureus infection and/or the S. aureusassociated condition in the subject.

In accordance with this aspect of the invention, suitable inhibitors ofLukE/D mediated cytotoxicity include protein or peptide inhibitors,nucleic acid inhibitors, or small molecule inhibitors.

In one embodiment of the invention, the inhibitor of LukE/D mediatedcytotoxicity is a LukE inhibitor. Suitable LukE inhibitors includeantibodies or antibody fragments recognizing an epitope in the aminoacid sequence of SEQ ID NO:11. In another embodiment of the invention,the inhibitor of LukE/D mediated cytotoxicity is a LukD inhibitor.Suitable LukD inhibitors include antibodies or antibody fragmentsrecognizing an epitope in the amino acid sequence of SEQ ID NO:22.

In another embodiment of this aspect of the present invention, theinhibitor of LukE/D mediated cytotoxicity inhibits LukE and LukDinteraction. Suitable inhibitors in accordance with this embodimentinclude anti-LukE and/or LukD antibodies that target the interactingregions of LukE or LukD. Alternatively, suitable inhibitors includesmall molecules that bind to the interacting regions of LukE and/orLukD. These interacting regions may include amino acids 3-13 of SEQ IDNO:11, amino acids 32-47 of SEQ ID NO:11, amino acids 126-139 of SEQ IDNO:11, amino acids 151-156 of SEQ ID NO:11, and amino acids 272-283 ofSEQ ID NO:11. The interacting regions may also include amino acids: 3-17of SEQ ID NO:22, amino acids 33-51 of SEQ ID NO:22, amino acids 94-113of SEQ ID NO:22, amino acids 115-131 of SEQ ID NO:22, and amino acids229-2741 of SEQ ID NO:22.

In another embodiment of this aspect of the present invention, theinhibitor of LukE/D mediated cytotoxicity inhibits LukE/D from bindingto the plasma membrane of leukocytes. Suitable inhibitors includeantibodies or small molecules recognizing the epitopes of LukE and/orLukD that interact with the plasma membrane of leukocytes. The regionsof LukE and LukD that interact with the plasma membrane include theamino acids encompassing the rim domain of LukE. These amino acidregions include LukE amino acids 57-75 of SEQ ID NO:11, amino acids82-99 of SEQ ID NO:11, amino acids 162-198 of SEQ ID NO:11, amino acids190-235 of SEQ ID NO:11, amino acids 263-284 of SEQ ID NO:11, and aminoacids 230-273 of SEQ ID NO:11 and LukD amino acids 59-75 of SEQ IDNO:22, amino acids 170-220 of SEQ ID NO:22, and amino acids 253-268 ofSEQ ID NO:22. Accordingly, antibodies recognizing these epitopes of LukEand/or LukD are particularly suitable for this embodiment of theinvention.

In another embodiment of this aspect of the present invention, theinhibitor of LukE/D mediated cytotoxicity is an agent that preventsLukE/D oligomer complex formation, an agent that blocks LukE/LukDmediated pore formation, or an agent that blocks the LukE/LukD pore. Inaccordance with this embodiment, suitable inhibitors of the LukE/LukDmediated pore include cyclodextrin and related compounds, and any otherpore inhibitor including protein or peptide inhibitors, nucleic acidinhibitors, or small molecule inhibitors.

In yet another embodiment of this aspect of the present invention, theinhibitor of LukE/D mediated cytotoxicity is an agent that modulates theexpression and/or activity of an endogenous repressor or activator ofLukE/D expression. Accordingly, administering an agent that induces ormimics the expression and or activity of Repressor of Toxins (“Rot”),which is a repressor of lukE and lukD expression, inhibits LukE/Dmediated cytotoxicity by virtue of blocking toxin production. Suitableagents that mimic Rot expression and activity and are suitable for usein the methods of the present invention are disclosed in U.S. PatentApplication Publication No. 2003/0171563 to McNamara, which is herebyincorporated by reference in its entirety. Likewise, administering anagent that inhibits the expression or activity of SaeRS, which is anactivator of lukE and lukD expression, inhibits LukE/D mediatedcytotoxicity by virtue of blocking toxin production.

For purposes of this and other aspects of the invention, the target“subject” encompasses any animal, preferably a mammal, more preferably ahuman. In the context of administering a composition of the inventionfor purposes of preventing a S. aureus infection in a subject, thetarget subject encompasses any subject that is at risk of being infectedby S. aureus. Particularly susceptible subjects include infants andjuveniles, as well as immunocompromised juvenile, adults, and elderlyadults. However, any infant, juvenile, adult, or elderly adult orimmunocompromised individual at risk for S. aureus infection can betreated in accordance with the methods of the present invention. In thecontext of administering a composition of the invention for purposes oftreating a S. aureus infection in a subject, the target subjectpopulation encompasses any subject infected with S. aureus. Particularlysuitable subjects include those at risk of infection or those infectedwith methicillin-resistant S. aureus (MRSA) or methicillin sensitive S.aureus (MSSA).

In the context of using therapeutic compositions of the presentinvention to prevent a S. aureus infection, either via active or passivevaccination, the concentration of LukE and LukD proteins or polypeptidesor anti-LukE and anti-LukD antibodies in the composition are adequate toachieve the prevention of S. aureus infection, particularly theprevention of S. aureus in susceptible populations. In the context ofusing therapeutic compositions to treat a S. aureus infection, theamounts of anti-LukE and anti-LukD antibodies or agents that inhibitLukE/D mediated cytotoxicity are capable of achieving a reduction in anumber of symptoms, a decrease in the severity of at least one symptom,or a delay in the further progression of at least one symptom, or even atotal alleviation of the infection.

Therapeutically effective amounts of LukE, LukD, anti-LukE and anti-LukDantibodies, and agents that inhibit LukE/D mediated cytotoxicity can bedetermined in accordance with standard procedures, which take numerousfactors into account, including, for example, the concentrations ofthese active agents in the composition, the mode and frequency ofadministration, the severity of the S. aureus infection to be treated(or prevented), and subject details, such as age, weight and overallhealth and immune condition. General guidance can be found, for example,in the publications of the International Conference on Harmonization andin REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990),which is hereby incorporated by reference in its entirety. A clinicianmay administer LukE and LukD or anti-LukE and anti-LukD antibodies,until a dosage is reached that provides the desired or requiredprophylactic or therapeutic effect. The progress of this therapy can beeasily monitored by conventional assays.

Therapeutically effective amounts of LukE and LukD for immunization willdepend on whether adjuvant is co-administered, with higher dosages beingrequired in the absence of adjuvant. The amount of LukE and LukD foradministration sometimes varies from 1 μg-500 μs per patient and moreusually from 5-500 μg per injection for human administration.Occasionally, a higher dose of 1-2 mg per injection is used. Typicallyabout 10, 20, 50 or 100 μg is used for each human injection. Preferably,the amounts of LukE and LukD are substantially the same. The timing ofinjections can vary significantly from once a day, to once a year, toonce a decade. Generally an effective dosage can be monitored byobtaining a fluid sample from the subject, generally a blood serumsample, and determining the titer of antibody developed against the LukEand LukD protein or polypeptide, using methods well known in the art andreadily adaptable to the specific antigen to be measured. Ideally, asample is taken prior to initial dosing and subsequent samples are takenand titered after each immunization. Generally, a dose or dosingschedule which provides a detectable titer at least four times greaterthan control or “background” levels at a serum dilution of 1:100 isdesirable, where background is defined relative to a control serum orrelative to a plate background in ELISA assays.

Therapeutically effective amount of the LukE and LukD antibodycompositions typically are at least 50 mg composition per kilogram ofbody weight (mg/kg), including at least 100 mg/kg, at least 150 mg/kg,at least 200 mg/kg, at least 250 mg/kg, at least 500 mg/kg, at least 750mg/kg and at least 1000 mg/kg, per dose or on a daily basis. Dosages formonoclonal antibody compositions might tend to be lower, such as aboutone-tenth of non-monoclonal antibody compositions, such as at leastabout 5 mg/kg, at least about 10 mg/kg, at least about 15 mg/kg, atleast about 20 mg/kg, or at least about 25 mg/kg. In some methods, twoor more monoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated. Antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody in the subject.Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe subject. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the subject shows partial orcomplete amelioration of symptoms of disease.

The therapeutic compositions of the present invention can beadministered as part of a combination therapy in conjunction withanother active agent, depending upon the nature of the S. aureusinfection that is being treated. Such additional active agents includeanti-infective agents, antibiotic agents, and antimicrobial agents.Representative anti-infective agents that may be useful in the presentinvention include vancomycin and lysostaphin. Representative antibioticagents and antimicrobial agents that may be useful in the presentinvention include penicillinase-resistant penicillins, cephalosporinsand carbapenems, including vancomycin, lysostaphin, penicillin G,ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin,cephalothin, cefazolin, cephalexin, cephradine, cefamandole, cefoxitin,imipenem, meropenem, gentamycin, teicoplanin, lincomycin andclindamycin. Dosages of these antibiotics are well known in the art.See, e.g., MERCK MANUAL OF DIAGNOSIS AND THERAPY, Section 13, Ch. 157,100^(th) Ed. (Beers & Berkow, eds., 2004), which is hereby incorporatedby reference in its entirety. The anti-inflammatory, anti-infective,antibiotic and/or antimicrobial agents may be combined prior toadministration, or administered concurrently (as part of the samecomposition or by way of a different composition) or sequentially withthe inventive therapeutic compositions of the present invention. Incertain embodiments, the administering is repeated. The subject may bean infant, juvenile, adult, or elderly adult. The subject may also be animmuno-compromised juvenile, adult, or elderly adult.

Therapeutic compositions of the present invention may be administered ina single dose, or in accordance with a multi-dosing protocol. Forexample, relatively few doses of the therapeutic composition areadministered, such as one or two doses. In embodiments that includeconventional antibiotic therapy, which generally involves multiple dosesover a period of days or weeks, the antibiotics can be taken one, two orthree or more times daily for a period of time, such as for at least 5days, 10 days or even 14 or more days, while the antibody composition isusually administered only once or twice. However, the different dosages,timing of dosages and relative amounts of the therapeutic compositionand antibiotics can be selected and adjusted by one of ordinary skill inthe art.

Compositions for of the present invention can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraperitoneal,intranasal or intramuscular means for prophylactic and/or therapeutictreatment. The most typical route of administration is subcutaneousalthough others can be equally effective. The next most common isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. Intravenous injections as well asintraperitoneal injections, intra-arterial, intracranial, or intradermalinjections are also effective in generating an immune response.

The pharmaceutical agents of the present invention may be formulated forparenteral administration. Solutions or suspensions of the agent can beprepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. Illustrativeoils are those of petroleum, animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, or mineral oil. In general, water,saline, aqueous dextrose and related sugar solution, and glycols, suchas propylene glycol or polyethylene glycol, are preferred liquidcarriers, particularly for injectable solutions. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

Pharmaceutical formulations suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

When it is desirable to deliver the pharmaceutical agents of the presentinvention systemically, they may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Intraperitoneal or intrathecal administration of the agents of thepresent invention can also be achieved using infusion pump devices suchas those described by Medtronic, Northridge, Calif. Such devices allowcontinuous infusion of desired compounds avoiding multiple injectionsand multiple manipulations.

In addition to the formulations described previously, the agents mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

A further aspect of the present invention relates to a method ofpredicting severity of an S. aureus infection. This method involvesculturing S. aureus obtained from an infected subject via a fluid ortissue sample from the subject and quantifying LukE and/or LukDexpression in the cultured S. aureus. The quantified amounts of LukEand/or LukD in the sample from the subject are compared to the amount ofLukE and/or LukD in a control sample which produces little orundetectable amounts of LukE and/or LukD and the severity of the S.aureus infection is predicted based on said comparing.

Another aspect of the present invention relates to a method of treatinga subject with a S. aureus infection. This method involves culturing S.aureus obtained from an infected subject via a fluid or tissue samplefrom the subject and quantifying LukE and/or LukD expression in thecultured S. aureus. The quantified amounts of LukE and/or LukD in thesample from the subject are compared to the amount of LukE and/or LukDin a control sample which produces little or undetectable amounts ofLukE and/or LukD, and a suitable treatment for the subject is determinedbased on this comparison. The method further involves administering thedetermined suitable treatment to the subject to treat the S. aureusinfection.

In accordance with these aspects of the invention, quantifying LukE andLukD expression in the sample from the subject involves measuring thelevel of LukE and/or LukD mRNA expression or protein production. Methodsof quantifying mRNA and protein expression levels in a sample are wellknown in the art. An increased level of LukE and/or LukD mRNA expressionor protein production in the sample from the subject compared to thecontrol sample indicates or predicts the subject has or will have a moresevere S. aureus infection. Likewise, an increased level of LukE and/orLukD mRNA expression or protein production in the sample from thesubject indicates that a suitable treatment for the subject having theinfection involves one or more agents that inhibit LukE/D mediatedcytotoxicity. Suitable agents for inhibiting LukE/D cytotoxicity arediscloses supra.

Another aspect of the present invention relates to a method ofidentifying inhibitors of LukE/D cytotoxicity. This method involvesproviding a cell population, a preparation containing LukE and LukD, anda candidate LukE/D inhibitor. The cell population is exposed to thepreparation containing LukE and LukD in the presence and absence of thecandidate inhibitor, and LukE/D mediated cytotoxicity is measured in thepresence and in the absence of the candidate inhibitor. The measuredamount of cytotoxicity in the presence and in the absence of thecandidate inhibitor is compared and an inhibitor of LukE/D cytotoxicityis identified based on this comparison.

In accordance with this aspect of the invention, anti-LukE and anti-LukDantibodies, and fragments thereof, as well as other potentialtherapeutic moieties (e.g., small organic molecules) may be screened toevaluate their ability to inhibit LukE/D mediated cytotoxicity. Asdescribed below, various methods have been designed to identify agentsthat inhibit some aspect of the cascade of events that leads to LukE/Dmediated cytotoxicity and lysis of human leukocytes. The methods arealso designed to identify altered forms of LukE and LukD that possessreduced toxicity relative to their native counterparts. The targetedevents that are part of the cascade include for example, binding of LukEand/or LukD to leukocyte plasma membranes, interaction between LukE andLukD (LukE/D oligomerization), and blockage of the membrane pore formedby the LukE/D oligomer. The assay formats generally require humanleukocytes (or LukE or LukD membrane-binding portion thereof), suitableculture medium, and purified LukE and LukD.

A person of skill will appreciate that the following protocols aremerely illustrative and that various operating parameters such asreaction conditions, choice of detectable label and apparati (e.g.,instrumention for detection and quantification) may be varied as deemedappropriate.

The following methods are generally directed to identifying agents thatinhibit LukE/D cytotoxicity, without necessarily revealing the exactevent in the cascade that is affected.

To identify inhibitors of LukE/D cytotoxicity, human leukocytes (e.g.,THP-1) may be plated in 384-well clear-bottom black tissue culturetreated plate (Corning) at 5×10³ cells/well in a final volume of 50 μlof RPMI (Gibco) supplemented with 10% of heat inactivated fetal bovineserum (FBS). Cells may then be contacted/mixed/reacted/treated with thetest compound/molecule (˜5 μl/different concentrations) and thenintoxicated with LukE and LukD, which in preferred embodiments aresubstantially purified (5 μl of a ˜0.01-5 μM solution), preferably addedtogether, under culture conditions to allow for intoxication of theleukocytes by LukE and LukD, e.g., for 1 hr at 37° C., 5% CO₂. Ascontrols, cells may be treated with culture medium (100% viable) andwith 0.1% v/v Triton X100 (100% death).

In these embodiments, cells treated as described above may then beincubated with a dye to monitor cell viability such as CellTiter(Promega) (which enables determination of cell viability via absorbanceby measuring the number of viable cells in a culture by quantificationof the metabolic activity of the cells) and incubated for an additionaltime period (e.g., about 2 hrs at 37° C., 5% CO₂). Cell viability maythen be determined such as by measuring the colorimetric reaction at 492nm using a plate reader e.g., Envision 2103 Multi-label Reader(Perkin-Elmer). Percent viable cells may be calculated such as by usingthe following equation: % Viability=100×[(Ab₄₉₂ Sample−Ab₄₉₂TritonX)/(Ab₄₉₂ Tissue culture media). An increase in the 100% viabilitysuggests inhibition of LukE/D mediated cytotoxicity.

A variation of this assay is referred to as a membrane damage assay. Inthese embodiments, cells treated as described above (e.g., up to andincluding treating of the cells with test compound/molecule and thenintoxicating the cells with purified LukE and LukD), may then beincubated with a cell-impermeable fluorescent dye such as SYTOX green(0.1 μM; Invitrogen) (in accordance with manufacturer's instructions)and incubated e.g., for an additional 15 minutes at room temperature inthe dark. Fluorescence, as an indicator of membrane damage, may then bemeasured using a plate reader such as Envision 2103 Multilabel Reader(Perkin-Elmer) at Excitation 485 nm, Emission 535 nm. A decrease influorescence suggests inhibition of LukE/D cytotoxicity.

In another variation of this assay, cells treated as described above(e.g., up to and including treating of the cells with test compound andthen intoxicating the cells with purified LukE and LukD), may then beincubated with a marker of cell lysis and incubated for an additionalperiod of time at room temperature in the dark. The marker of cell lysisis measured, and a decrease in the level of cell lysis in the presenceof the compound indicates inhibition of LukE/D cytotoxicity.

Together these assays will facilitate the identification of compoundsthat inhibit or reduce LukE/D cytotoxic effects towards leukocyte cells.

Additional methods may be used, independently or in conjunction with themethods described above, particularly if the above methods revealinhibitory activity, that will enable a person skilled in the field todetermine more precisely what event in the biochemical cascade is beingaffected or targeted by the agent. These events include binding of LukEand/or LukD to leukocyte membranes, binding of LukE to LukD (LukE/Doligomerization), and blockage of the membrane pore formed by the LukE/Doligomers.

Another aspect of the present invention is directed to a method ofidentifying inhibitors of LukE, LukD, and/or LukE/D binding to targetcells. This method involves providing a population of leukocytes orother target cells, a preparation containing a detectably labeled LukE,LukD, or LukE/D, and a candidate inhibitor. The cell population isexposed to the preparation containing the detectably labeled LukE, LukD,or LukE/D in the presence and absence of the candidate inhibitor, andlabeled LukE, LukD, or LukE/D binding to the leukocyte population ismeasured in the presence and absence of the candidate inhibitor. Themeasured amount of LukE, LukD, or LukE/D binding in the presence and inthe absence of the candidate inhibitor is compared and an inhibitor ofLukE, LukD, or LukE/D-leukocyte binding is identified based on thiscomparison.

To screen for inhibitors that block or reduce LukE, LukD or LukE/Dbinding to target cells, which is the first step in the intoxicationprocess, human leukocytes (e.g., THP-1 cells) may be plated in 384-wellflat-bottom tissue culture treated plates (Corning) at 2.5×10³cells/well in a final volume of 50 μl of RPMI (Gibco) supplemented with10% of heat inactivated fetal bovine serum (FBS). Cells may then betreated with the test compound/molecule (˜5 μl/different concentrations)and incubated with purified, fluorescently labeled LukE, LukD and/orLukE/D (e.g., FITC, Cy3, Cy5, APC, PE) 5 μl of a ˜0.01-5 μM solution for1 hr at 4° C., 5% CO₂. To evaluate the efficacy of the testedcompounds/molecules, the cell-associated fluorescence may be measured asan indicator of LukE, LukD, or LukE/D binding to cells e.g., using anautomated fluorescence microscopic imaging system designed for highcontent screening and high content analysis (e.g., Cellomics ArrayScanHCS Reader (Thermo Scientific) (Excitation 485 nm, Emission 535 nm)). Inaccordance with this aspect of the invention, a decrease in LukE, LukD,or LukE/D-leukocyte binding in the presence of the candidate inhibitorcompared to in its absence identifies a binding inhibitor.

To screen for inhibitors that block or reduce LukE/LukD interaction,which is the second step in the intoxication process, human leukocytes(e.g., THP-1 cells) may be plated in 384-well flat-bottom tissue culturetreated plates (Corning) at 2.5×10³ cells/well in a final volume of 50μl of RPMI (Gibco) supplemented with 10% of heat inactivated fetalbovine serum (FBS). Cells may then be treated with the testcompound/molecule and then intoxicated with a mixture of purified LukEand purified LukD where LukD is fluorescently-labeled with afluorescence molecule such as FITC, Cy3, Cy5, APC, and PE, and allowedto stand to complete the intoxication process (e.g., for 1 hr at 37° C.,5% CO₂). To evaluate the efficacy of the tested compounds/molecules,cell-associated LukD-FITC fluorescence may be measured as an indicatorof LukE/LukD-FITC interaction, using for example, an automatedfluorescence microscopic imaging system designed for high contentscreening and high content analysis (e.g., a Cellomics ArrayScan HCSReader (Thermo Scientific) (Excitation 485 nm, Emission 535 nm). Similarexperiments could be performed using fluorescently-labeled LukE insteadof LukD.

Another aspect of the present invention relates to a method ofidentifying inhibitors of LukE/D mediated pore formation. This methodinvolves providing a population of leukocytes, a preparation containingLukE and LukD, and a candidate inhibitor. The leukocyte population isexposed to the preparation containing LukE and LukD in the presence andabsence of the candidate inhibitor, and pore formation on the leukocytepopulation is measured in the presence and absence of the candidateinhibitor. The measured amount of pore formation in the presence and inthe absence of the candidate inhibitor is compared, and an inhibitor ofLukE/D mediated pore formation is identified based on that comparison.

To screen for inhibitors that block or inhibit formation of the LukE/Dpore, the effector molecule that leads to cell lysis, human leukocytes(e.g., THP-1 cells) may be plated in 384-well clear-bottom black tissueculture treated plate (Corning) at 2.5×10³ cells/well in a final volumeof 50 μl of RPMI (Gibco) supplemented with 10% of heat inactivated fetalbovine serum (FBS). Cells may then be treated with the testcompound/molecule (˜5 μl containing different concentrations) and thenintoxicated with purified LukE and LukD or LukE/D (˜0.01-5 μM) for 15minutes at 37° C., 5% CO₂. As controls, cells may be treated withculture medium (negative control) and with 0.1% v/v TritonX100 (positivecontrol).

To directly evaluate LukE/D pores on the surface of host cells, anethidium bromide (EB) influx assay may be used. EB is a small-cationicdye that is impermeable to healthy host cells. Upon formation ofcationic pores by LukE/D, EB enters the cells and binds to DNA, whichresults in fluorescence. Cell treated in this fashion may then beincubated with EB (5 μM) for an additional 5 minutes at room temperaturein the dark. To evaluate the efficacy of the tested compounds/moleculesin inhibiting LukE/D pore formation the fluorescence may be measured asan indicator of pore formation, using a plate-reader such as theEnvision 2103 Multilabel Reader (Perkin-Elmer) at Excitation 530 nm,Emission 590 nm. This assay will facilitate the identification ofmolecules that can block or inhibit the LukE/D pore, which willalleviate LukE/D mediated toxicity.

To directly evaluate LukE/D pores on the surface of host cells, anethidium bromide (EB) influx assay may be used. EB is a small-cationicdye that is impermeable into healthy host cells. Upon formation ofcationic pores by LukE/D, EB enters the cells and binds to DNA, whichresults in fluorescence (see e.g., FIG. 5E). Cells treated with an agentcausing LukE/D pore formation may then be incubated with EB (5 μM) foran additional 5 minutes at room temperature in the dark. To evaluate theefficacy of the tested compounds/molecules in inhibiting LukE/D poreformation the fluorescence may be measured as an indicator of poreformation, using a plate-reader such as the Envision 2103 MultilabelReader (Perkin-Elmer) at Excitation 530 nm, Emission 590 nm. This assaywill facilitate the identification of molecules that can block orinhibit the LukE/D pore, which will alleviate LukE/D mediated toxicity.

The candidate compounds utilized in the assays described herein may beessentially any compound or composition suspected of being capable ofaffecting biological functions or interactions. The compound orcomposition may be part of a library of compounds or compositions.Alternatively, the compound or compositions may be designed specificallyto interact or interfere with the biological activity of LukE, LukD, orLukE/D of the present invention.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1—Inactivation of Rot Enhances the Virulence of a S. aureusStrain Lacking Agr

In recent studies it has been found that S. aureus mutant strains thatlack both the master regulator known as the accessory gene regulator(“Agr”) and the transcription factor repressor of toxins (“Rot”) (i.e.,Δag Δrrot) exhibit enhanced virulence in a murine model of systemicinfection compared to the highly attenuated Δagr mutant. While a Δagrsingle deletion mutant is highly attenuated for virulence as measured bysurvival over time post-infection, an Δagr Δrot double mutant displaysvirulence characteristics similar to that of the parent strain (WTNewman) (FIG. 1A). It was speculated that the increased virulenceobserved in an Δagr Δrot double mutant might be due to enhancedexpression of S. aureus leukotoxins as many of these toxins are believedto be regulated in an Agr-Rot dependent manner. Indeed, immunoblotanalysis of the toxins produced by S. aureus Wild Type, Δagr, and theΔagr Δrot mutant strains confirmed the hypothesis as a number of toxinswere restored to WT levels in an Δagr Δrot double mutant (FIG. 1B).Strikingly, it was observed that LukE/D is highly over-produced by theΔagr Δrot strain compared to the other toxins (FIG. 1B). This datademonstrates that repression of key virulence factors by Rot is criticalto optimal virulence potential in S. aureus and that the leukotoxinLukE/D is heavily repressed in a Rot-dependent manner, more so thanother leukotoxins.

Example 2—LukE/D Contributes to the Enhanced Virulence Exhibited by a S.aureus Strain Lacking Rot

The results described in FIGS. 1A-1B indicated that inactivation of rotin an agr⁺ strain might also result in increased virulence, possibly ina LukE/D dependent manner. As with the Δagr Δrot double mutant (FIG.1A), it was observed that a Δrot single deletion mutant also resulted inenhanced virulence in a murine model of systemic infection, as evidencedby the decrease in percent survival of animals infected with Δrotcompared to those infected with WT (FIG. 2A). Earlier observationsdemonstrated that Rot is likely a major repressor of the leukotoxinLukE/D. To confirm these findings in the context of the single Δrotdeletion mutant, immunoblot were performed. These experiments revealedthat indeed LukE/D is highly produced in the absence of rot, contrary toLukAB, γ-hemolysin (HlgC), or α-toxin (Hla; FIG. 2B). These findingsfurther strengthened the supposition that LukE/D is the majorRot-repressed factor responsible for the increase in virulence of a Δrotmutant and that LukE/D could play a significant role in the in vivopathogenesis of S. aureus. To determine whether LukE/D overproductionwas responsible for the enhanced pathogenic phenotype of Δrot, a ΔrotΔlukE/D double mutant was constructed and its virulence in a mouse modelof infection was assessed. The Δrot ΔlukE/D double mutant wassignificantly compromised for virulence as evidenced by the strikingreduction in mortality compared to both WT as well as the Δrot mutant(FIG. 2C). These results demonstrate that LukE/D is a majorRot-repressed factor that is critical for the hypervirulence associatedwith a Δrot mutant and that LukE/D may be a major contributor to diseasein general. These data further indicate that drugs that enhance Rotmediated repression of target genes will reduce S. aureus pathogenesis.

Example 3—Rot Represses LukE/D Expression by Directly Binding to theLukE/D Promoter

To further examine the influence of Rot on lukE/D gene expression,transcriptional fusions of the lukE/D promoter region to a gene thatencodes for the green fluorescent protein (GFP) were constructed andfluorescence was measured over time in broth culture using WT, Δagr,Δrot, and Δagr Δrot strains. As suspected, gene expression of lukE/D wasincreased over that of WT in strains lacking Rot, while strainsexpressing large amounts of Rot (Δagr mutants display increased Rotlevels) were decreased in expression. To assess whether repression oflukE/D by Rot is direct, the ability of Rot to bind to the lukE/Dpromoter was examined using a promoter pull-down strategy. Bacterialwhole cell lysates were incubated with lukE/D promoter DNA ornonspecific intergenic DNA bound to magnetic beads. Immunoblot of boundproteins demonstrated that Rot indeed binds to the lukE/D promoter in aspecific manner (FIG. 3B). These results implicate Rot as a directrepressor of lukE/D gene expression. Alterations in Rot levels couldthus substantially increase or decrease the production of LukE/D and byconsequence modulate the virulence potential of S. aureus.

Example 4—LukE/D Significantly Contributes to S. aureus Pathogenesis

Not only does a Δrot ΔlukE/D double deletion mutant eliminate thehypervirulence associated with a rot deletion, it also substantiallyreduces virulence overall (compare WT survival to Δrot ΔlukE/D survivalFIG. 3B). To test whether LukE/D plays a major role in the pathogenesisof S. aureus septicemic infection, a ΔlukE/D mutant in the strain Newmanwas constructed (FIGS. 4A and 4B) and the impact of lukE/D deletionalone on virulence was examined. Survival over time dramaticallyincreased for mice infected with either 10⁷ or 10⁸ CFU of the ΔlukE/Dmutant compared to the wild type. All wild type mice succumbed toinfection by 250 hours at the 10⁷ dose (FIG. 4C) and by 100 hours at the10⁸ dose (FIG. 4D). In both cases however, nearly 100% of mice infectedwith ΔlukE/D mutant survived until at least 300 hours post infection, aphenotype that is fully complemented with the ΔlukE/D::plukE/D strain(FIGS. 4B and 4C). In addition, bacterial burden to the kidney isreduced by 10-fold compared to the wild type or complemented strains(FIG. 4E) and abscess formation is significantly reduced (FIG. 4F).These results show that LukE/D is indeed a critical virulence factor forS. aureus systemic infection. Thus LukE/D is an attractive novel targetfor development of new therapeutics to counter S. aureus infection.

Example 5—LukE/D Targets and Kills Leukocytes

To determine the potential mechanism of action of LukE/D, recombinantLukE and LukD proteins from E. coli were expressed and purified. To testthe potential toxicity of these proteins, human monocyte-like cells(THP-1s) and human promyelocytic leukemia cells (HL60s) were incubatedwith different concentrations of either individual subunits (i.e., LukEor LukD) or a mixture of LukE+LukD (LukE/D). Cells were intoxicated witha dose response of either LukE alone, LukD alone, or LukE/D and after 1hour of intoxication CellTiter was added to measure the percent viablecells post-intoxication. The human monocyte-like cell line, THP-1, wasuniquely sensitive to intoxication with both subunits of the toxintogether but not individual subunits. The potency of the toxin towardsthe cells was dose dependent and strictly required the presence of bothsubunits (FIG. 5A). In contrast, HL60s were not affected by eithersubunit alone or incubated together (FIG. 5B). In addition to celllines, the activity of LukE/D towards primary human and murine PMNs wasalso examined. Cells were intoxicated with a dose response of eitherLukE alone, LukD alone, or LukE/D and after 1 hour of intoxicationCellTiter was added to measure the percent viable cellspost-intoxication. LukE/D but not LukE or LukD was markedly cytotoxictowards PMNs from both human and mouse (FIG. 5C).

To examine the mechanism by which LukE/D is toxic to THP-1s, cells wereintoxicated in the presence of ethidium bromide, a small cationic dyethat is normally impermeable to host cell membranes, but can gain accessto the cell via the toxin pore. Upon addition of both toxin subunits,ethidium bromide was rapidly taken up into cells as reflected by anincrease in relative fluorescence compared to unintoxicated controls andintoxicated PMN-HL60s (FIGS. 5D and 5E). These experiments demonstratethat when together, LukE and LukD exhibit cytotoxicity toward specifichuman immune cell type, but not others, highlighting the specificity ofthis toxin.

Example 6—Antibodies Against LukE Potently Neutralized LukE/DCytotoxicity

To determine if polyclonal antibodies raised against LukE are capable ofneutralizing LukE/D cytotoxicity, a neutralization assay was performed.Incubation of LukE/D with purified anti-LukE antibodies potentlyinhibited LukE/D mediated cytotoxicity towards THP-1 cells as measuredby CellTiter (FIG. 6A). As shown in FIGS. 5A-5E, LukE/D appears to exertits toxic activity by forming permeable pores in the plasma membrane oftarget cells. To gain insight into the mechanism by which anti-LukEneutralizes LukE/D cytotoxicity, the formation of LukE/D pores in cellsintoxicated with LukE/D in the presence of purified anti-LukE antibodieswas monitored. It was observed that LukE/D pore formation was potentlyinhibited by the anti-LukE antibody (FIG. 6B). These data demonstratethat immunization with LukE generates anti-LukE neutralizing antibodies,suggesting LukE-specific antibodies could be a novel therapeutic tocombat S. aureus infection.

Although the invention has been described in detail for the purposes ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. (canceled)
 2. A composition for immunizing a subject against S.aureus infection, said composition comprising: an isolated Leukocidin E(LukE) polypeptide, said polypeptide comprising an amino acid sequenceof 50-200 amino acid residues of SEQ ID NO: 11; and a pharmaceuticallyacceptable carrier.
 3. The composition of claim 2 further comprising: anisolated Leukocidin D (LukD) polypeptide, said polypeptide comprising anamino acid sequence of 50-200 amino acid residues of SEQ ID NO:
 22. 4.The composition of claim 2 further comprising: an adjuvant.
 5. Thecomposition of claim 4, wherein the adjuvant is selected from the groupconsisting of flagellin, Freund's complete adjuvant, Freund's incompleteadjuvant, aluminum hydroxide, lysolecithin, pluronic polyols,polyanions, peptides, oil emulsion, dinitrophenol, iscomatrix, andliposome polycation DNA particles.
 6. A composition for immunizing asubject against S. aureus infection, said composition comprising: anisolated Leukocidin E (LukE) polypeptide comprising an amino acidsequence having at least 80% sequence identity to the sequence of aminoacid residues 29-311 of SEQ ID NO: 11; and a pharmaceutically acceptablecarrier.
 7. The composition of claim 6, wherein the amino acid sequenceof the isolated LukE polypeptide comprises at least 90% sequenceidentity to the sequence of amino acid residues 29-311 of SEQ ID NO: 11.8. The composition of claim 6, wherein the amino acid sequence of theisolated LukE polypeptide comprises at least 95% sequence identity tothe sequence of amino acid residues 29-311 of SEQ ID NO:
 11. 9. Thecomposition of claim 6 further comprising an isolated Leukocidin D(LukD) polypeptide, said polypeptide comprising an amino acid sequenceof 50-200 amino acid residues of SEQ ID NO:
 22. 10. The composition ofclaim 6 further comprising: an adjuvant.
 11. The composition of claim10, wherein the adjuvant is selected from the group consisting offlagellin, Freund's complete adjuvant, Freund's incomplete adjuvant,aluminum hydroxide, lysolecithin, pluronic polyols, polyanions,peptides, oil emulsion, dinitrophenol, iscomatrix, and liposomepolycation DNA particles.
 12. A composition for immunizing a subjectagainst S. aureus infection, said composition comprising: an isolatedLeukocidin D (LukD) polypeptide, said polypeptide comprising an aminoacid sequence of 50-200 amino acid residues of SEQ ID NO: 22; and apharmaceutically acceptable carrier.
 13. The composition of claim 12further comprising an isolated Leukocidin E (LukE) polypeptide, saidpolypeptide comprising an amino acid sequence of 50-200 amino acidresidues of SEQ ID NO:
 11. 14. The composition of claim 12 furthercomprising: an adjuvant.
 15. The composition of claim 12, wherein theadjuvant is selected from the group consisting of flagellin, Freund'scomplete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide,lysolecithin, pluronic polyols, polyanions, peptides, oil emulsion,dinitrophenol, iscomatrix, and liposome polycation DNA particles.